The invention discloses a new type-1 ribosome-inactivating protein (RIP), referred to as bouganin, isolated from the leaves of Bougainvillea species, especially B. spectabilis Willd. Bouganin differs from other type-1 RIP by its unique amino acid composition. Bouganin has a molecular weight of about 26,200 daltons. Bouganin is useful as an immunomodulator, anti-viral agent or anti-tumour agent. Compositions comprising bouganin and a cell binding ligand are particularly useful to kill cells of a target population.
It has been known for a long time that extracts from many plant tissues possess anti-viral activity, which in several cases is due to proteins identified as inhibitors of protein synthesis, called ribosome-inactivating proteins (RIP, reviewed by Barbieri et al., Biochim. Biophys. Acta 1154:237 (1993)). The pokeweed anti-viral protein (PAP) was the first anti-viral protein to be identified as a RIP (reviewed by Irvin, in Antiviral Proteins in Higher Plants 65 (1994)). Subsequently, all other RIP tested possess anti-viral activity not only against plant viruses, but also against animal viruses, including HIV (reviewed by Battelli and Stirpe, in Antiviral Proteins in Higher Plants (1994)).
All RIP, either single-chain (type-1) or two-chain (type-2), enzymatically release adenine from a single nucleotide in a precise position (A4324 in the case of rat liver 28S rRNA, A2660 of E. coli rRNA) in a universally conserved GAGA tetraloop of the major rRNA (Endo and Tsurugi, J. Biol. Chem. 262:8128 (1987); reviewed by Barbieri et al., Biochim. Biophys. Acta 1154:237 (1993)). Depurinated ribosomes become unable to elongate the nascent peptide chain.
The anti-viral activity of these proteins was commonly attributed to the inactivation of ribosomes, with inhibition of protein synthesis of the host cell and consequent arrest of viral replication. However a degradation of supercoiled DNA in the presence of RIP was reported (Li et al., Nucleic Acid Res. 22:6309 (1991); Ling et al., FEBS Lett. 345:143 (1994); Roncuzzi and Gasperi-Campani, FEBS Let. 392:16 (1996)). Moreover, at least some RIP release more than one adenine residue from ribosomes (Barbieri et al., Biochem. J. 286:1 (1992)) and act on RNA species other than ribosomal, including viral RNAs, on poly(A), and on DNA (Barbieri et al., Nature 372.624 (1994), Nucleic Acid Res. 25:518 (1997); Stirpe et al., FEBS Lett. 382:309 (1996)). Thus many, if not all, RIP have polynucleotide:adenosine glycosidase activity, which may have a role in the anti-viral activity besides the inactivation of the host cell ribosomes.
Immunotoxins are chimeric molecules in which cell-binding ligands are coupled to toxins or their subunits. The ligand portion of the immunotoxin is usually an antibody that binds to selected target cells. The toxin portion of the immunotoxin can be derived form various sources. Most commonly, toxins are derived from plants or bacteria, but toxins of human origin or synthetic toxins (drugs) have been used as well. Toxins used for immunotoxins derived from plants or bacteria all inhibit protein synthesis of eukaryotic cells. The most widely used plant toxin, ricin, consist of two disulfide-linked polypeptides A and B (Olsnes et al., in Molecular Action of Toxins and Viruses 51 (1982)). Another group of plant-derived toxins used in immunotoxins are the type-1 RIP. These molecules are single-chain proteins found in plants and have similar enzymatic properties as the A-chain of ricin (reviewed in Stirpe and Barbieri FEBS Lett. 195:1 (1986)).
The cross-linker used to join the ligand (antibody) and the toxin must remain stable when extracellular, but labile when intracellular, so that the toxin fragment can enter the cytosol. The choice of cross-linker depends on whether intact toxins, A-chains or type-1 RIP are used. A-chains and type-1 RIP are generally coupled to the ligand using Linkers that introduce a disulfide bond between the ligand and the A-chain (Myers et al., J. Immunol. Meth. 136:221 (1991)). Intact toxins are usually linked to ligands using non-reducible linkages (such as thioether) to prevent release of the active free toxin in vivo. Recombinant immunotoxins have been prepared by splicing the genes encoding the toxin to the gene encoding the ligand (for instance a recombinant antibody fragment) and expressing the entire immunotoxin as a fusion protein (Pastan et al., Ann. Rev. Biochem. 61:331 (1992)). Recombinant immunotoxins are highly stable in vivo because they contain non-reducible peptide bonds.
Various types of immunotoxins directed against different cellular targets have been evaluated in vivo, both in animal models and in phase I or II clinical trials. The results of a number of these studies are reviewed in Ghetie and Vitetta Curr. Opin. Inmmunol. 6:707 (1994) and Thrush et al., Ann. Rev. Immunol. 14:49 (1996).
Ribosome inactivating proteins (RIP) comprise a class of proteins with potent inhibitory activity of eukaryotic protein synthesis. RIP can be classified in two groups. Type-1 RIP consist of a single peptide chain having ribosome inactivating activity, whereas type-2 RIP consist of an A chain with ribosome inactivating activity and a B chain having cell binding activity. Here we describe the isolation of a novel type-1 RIP, referred to as bouganin, with a low non-specific toxicity, making it very suitable for the incorporation as the toxin part in various immnunotoxin molecules. The invention pertains to this novel protein and biologically active peptide parts and equivalents thereof, to immunotoxins based On this protein, to the production of such proteins and immunotoxins, and to their use in the medical and plant-protection fields. The invention is defined in the appending claims.
The invention described herein draws on previously published work. By way of example, such work consists of scientific papers, patents and pending patent applications. All of these publications and applications, cited previously or below, are hereby incorporated by reference.
The protein according to the invention corresponds to the bouganin protein as described below in more detail, as well as to biologically active fragments and equivalents thereof. The term xe2x80x9cbiologically activexe2x80x9d means being capable of inhibiting protein synthesis in vitro or in vivo. Such fragments generally comprise one or more active sites of the protein or the encoding polynucleotide and generally comprise a sequence at least 8 amino acids, preferably at least 10, at least 15 or even at least 30 amino acids, of the protein, or the corresponding number of nucleotides of the polynucleotide.
The term xe2x80x9cligandxe2x80x9d refers to any molecule capable of binding with or otherwise recognizing a receptor on a target cell. The ligand may be a protein or a non-protein molecule. Examples of such ligands include, but are not limited to, antibodies, growth factors, cytokines, hormones and the like, that specifically bind desired target cells.
As used herein, the term xe2x80x9cimmunotoxinxe2x80x9d refers to chimeric molecules in which a cell binding ligand is coupled to the novel type-1 RIP bouganin or fragments thereof
As used herein, the term xe2x80x9cantibodyxe2x80x9d refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as. Fab, F(abxe2x80x2)2, Fv, and other fragments which retain the antigen binding function of the parent antibody.
As used herein, the term xe2x80x9cmonoclonal antibodyxe2x80x9d refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(abxe2x80x2)2, Fv, and others which retain the antigen binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in this invention. In practice, however, the antibodies will typically be of rat or murine origin because of the availability of rat or murine cell lines for use in making the required hybrid cell lines or hybridomas to produce monoclonal antibodies.
As used herein, the term xe2x80x9chumanized antibodiesxe2x80x9d means that at least a portion of the framework regions of an immunoglobulin are derived from human inmmunoglobulin sequences.
As used herein, the term xe2x80x9csingle chain antibodiesxe2x80x9d refer to antibodies prepared by determining the binding domains (both heavy and light chains) of a binding antibody, and supplying a linking moiety which permits preservation of the binding function. This forms, in essence, a radically abbreviated antibody, having only that part of the variable domain necessary for binding to the antigen. Determination and construction of single chain antibodies are described in U.S. Pat. No. 4,946,778 to Ladner et al. Methods for the generation of antibodies suitable for use in the present invention are well known to those skilled in the art and can be found described in such publications as Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).
The natural or recombinant type-1 RIP bouganin molecules of the present invention may be fused to, or otherwise bound to a ligand by any method known and available to those skilled in the art. The ligand and the bouganin molecules may be chemically bonded together by any of a variety of well-known chemical procedures, such as the use of hetero-bifunctional cross-linkers, e.g. SPDP, 2-iminothiolane, carbodiimide or glutaraldehyde. Production of various immunotoxins is well-known within the art and can be found, for example in Thorpe et al., Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet 168 (1982) and Waldmann, Science, 252:1657 (1991), both of which are incorporated by reference.
Bouganin may also be fused to the ligand by recombinant means such as through the production of single chain antibodies. The genes encoding ligand and bouganin may be cloned in cDNA form and linked directly or separated by a small peptide linker by any cloning procedure known to those skilled in the art. See for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, (1989).
A person skilled in the art will appreciate that additional modifications, deletions and insertions may be made to the ligand binding agent and bouganin genes. Especially, deletions or changes may be made in the linker connecting an antibody gene to bouganin. All such constructions may be made by methods of genetic engineering well known to those skilled in the art (see, generally, Sambrook et al., supra) and may produce proteins that have differing properties of affinity, specificity, stability and toxicity that make them particularly suitable for various clinical or biological applications.
Fusion proteins of the present invention including bouganin molecules may be expressed in a variety of host cells, including but not limited to bacterial hosts and yeast. The recombinant antibody-bouganin fusion protein gene will be linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal, The plasmids of the invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as, but not limited to, the ampiciline and neomycin genes.
Once expressed, the recombinant antibody-bouganin fusion proteins can be purified according to standard procedures of the art, such as described in R. Scopes, Protein Purification, Springer Verlag, N.Y. (1982). Substantially pure compositions of at least about 90 to 95% homogeneity are for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically.
The pharmaceutical compositions of this invention are administered at a concentration that is therapeutically effective to a patient in the need of a treatment. To accomplish this goal, the pharmaceutical compositions may be formulated using a variety of acceptable excipients known in the art. The compositions for administration will commonly comprise a solution of the bouganin molecule, antibody-bouganin conjugates, single chain antibody-bouganin fusion proteins, ligand-bouganin conjugates or single chain ligand-bouganin fusion proteins dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Typically, the compositions are administered by injection, either intravenously, intraperitoneally, in an other body cavity or into a lumen of an organ. Methods to accomplish this administration are known to those of ordinary skill in the art. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes.
The pharmaceutical compositions may also contain a polynucleotide encoding the bouganin molecule or active parts thereof, for use in gene therapy. The polynucleotide may be combined with means for specific delivery of the polynucleotide at the site were calls are to be killed by incorporation of the polynucleotide; such targeting means may e.g. be site-specific antibodies, liposomes or other art-known targeting devices.
Before administration to patients, formulants may be added to the antibodies. A liquid formulation is preferred. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as mono-, di-, or polysaccharides. The saccharides can include fructose, glucose, mannose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, xcex1- and xcex2-cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethyl cellulose, other water-soluble glucans, or mixtures thereof. Sucrose is most preferred. xe2x80x9cSugar alcoholxe2x80x9d is defined as a C4 to C8 hydrocarbon having OH groups, and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is most preferred. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably, the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v %. Preferably amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added. Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Any physiologically acceptable buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer. Preferably, the concentration is from 0.01 to 0.3 M. Surfactants can be added to the formulation, for example those shown in EP-A-270799 and EP-A-268110.
Additionally, antibody-bouganin conjugates or single chain antibody-bouganin fusion proteins can, for example, be chemically modified by covalent conjugation to a polymer to increase their circulating half-life. Preferred polymers, and methods to attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285 and 4,609,546, which are all hereby incorporated by reference in their entireties. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula R(Oxe2x80x94CH2xe2x80x94CH2)nOxe2x80x94R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40,000, more preferably between 2000 and 20,000, most preferably between 3,000 and 12,000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with a free amino group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention.
Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG has a preferred molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., J Biol. Chem. 263,15064 (1988), and a discussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106, both of which are hereby incorporated by reference in their entireties.
Another drug delivery system for increasing circulatory half-life is the liposome. Methods of preparing liposome delivery systems are discussed in Gabizon et al., Cancer Res. 42:4734 (1982); Cafiso, Biochim. Biophys. Acta 649:129 (1981); and Szoka, Ann. Rev. Biophys. Eng. 9:467 (1980). Other drug delivery systems are known in the art and are described in, e.g., Poznansky et al., Drug Delivery Systems 253 (1980), Poznansky, Pharm. Rev. 36:277 (1984).
After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer""s solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art. As stated above, the compositions of this invention are especially used to treat human patients. The preferred route of administraxtrose in saline. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.
The dosage and mode of administration will depend on the individual. Generally, the compositions are administered so that antibodies are given at a dose between 1 xcexcg/kg and 20 mg/kg, more preferably between 20 xcexcg/kg and 10 mg/kg. Preferably, it is given as a bolus. Continuous infusion may also be used, if so, the compositions may be infused at a dose between 1 and 100 xcexcg/kg/min.
The compositions containing the present pharmaceutical compositions or a cocktail thereof (i.e., with other pharmaceutically active proteins) can be administered for therapeutic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a xe2x80x9ctherapeutically effective dose.xe2x80x9d Amounts effective for this use will depend upon the severity of the disease and the general state of the patient""s health.
Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient.
Among various uses of the pharmaceutical compositions of the present invention are included a variety of disease conditions caused by specific human cells that may be eliminated by the toxic action of the protein. One preferred application is the treatment of cancer, such as by the use of a tumour cell binding antibody as the ligand or of autoimmune conditions such as graft-versus-host disease, organ transplant rejection, type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis and the like. The pharmaceutical compositions may also be used in vitro, for example, in the elimination of harmful cells from bone marrow before transplant. The ligand portion of bouganin containing conjugates and fusion proteins is chosen according to the intended use. A large number of cell membrane molecules on lymphocytes may serve as target of the ligand part of the immunotoxin. Also antigens found on cancer cells that may serve as targets for the ligand part of immunotoxin with bouganin. Those skilled in the art will understand that ligands may be chosen that bind to receptors expressed on other types of cells as well.
The bouganin molecule itself has also applications as an anti-viral compound. Type-1 RIP are know to be active against viruses affecting mammals and plants. Bouganin can therefore be used as a therapeutic molecule to treat viruses. The discovery of the anti-viral activity of RIP against a broad range of plant viruses when applied exogenous to inoculated leaves, has led to transfection of genes coding for RIP in host plants. Virus infection modifies the permeability of the cell membrane, thereby allowing the access of normally excluded molecules to the cytoplasm. RIP can then enter the virus infected cell and, once inside, inactivate ribosomes and viral replication. Besides the anti-viral activity of RIP, transfection of genes coding for RIP in host plant can also be applied to insect pest control. RIP are only moderately inhibitory for plant ribosomes but are highly inhibitory for ribosomes of plant parasites and are consequently good candidates for parasite control in plants. Transformation of an economically important host plant with the gene for a RIP which is toxic to parasites and is ineffective on the ribosomes of the plant confers specific resistance. An example of such a transgenic plant is a tobacco plant transfected with the Barley RIP. The constitutive expression of RIP in host plant can cause abnormal development of transgenic plant that can limit their application. To circumvent this problem a virus induced expression of RIP in transgenic plant is used, affecting only virus-infected cells without causing abnormal developing plants. Purified bouganin can also be applied directly in small amount on the leaves, completely preventing the mechanical transmission of unrelated viruses to several different host plants (Chen et al., Plant Pathol. 40:612 (1991)).
The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way.